The goal of this application is to develop a deeper understanding of how adhesive cues from the extracellular matrix (ECM) modulate endothelial cells' response to soluble growth factor signaling during angiogenesis, a process crucial to the vascularization of engineered and native infarcted tissues. Both soluble factors, such as vascular endothelial growth factor (VEGF), and cell-ECM adhesive interactions are known to regulate proliferation, migration, and capillary morphogenesis in angiogenesis. Cell adhesion involves binding of integrins to ECM ligands, as well as cell spreading against the substrate. While integrin binding effects on VEGF signaling are well studied, the effects of quantitative changes in cell spreading are not. We hypothesize that changes in cell spreading modulate VEGF signaling to regulate differential cell behaviors in angiogenesis and that our findings can be employed to improve efforts in vascularizing tissues. AIM 1: To investigate the effects of cell spreading on VEGF-induced endothelial cell proliferation and gene expression. The degree of cell-ECM adhesion will be quantitatively controlled by micropatterning of the adhesive ligand fibronectin, and we will study how these changes in spreading modulate VEGF-induced endothelial cell proliferation and gene expression, assessed by real-time PCR of representative genes determined previously by whole genome microarray analysis. AIM 2: To assess the effects of cell spreading on VEGF-induced ERK and RhoA signaling. The effect of spreading on two downstream pathways of VEGF signaling, ERK and RhoA, which we hypothesize to be critical mediators in VEGF-induced endothelial cell proliferation and gene expression, will be studied by ERK phosphorylation and RhoA G-LISA. AIM 3: To determine the roles of ERK and RhoA signaling in mediating cell spreading modulation of VEGF-induced proliferation and gene expression. Pharmacological and molecular interventions will be used to test the involvement of ERK and RhoA signaling in regulating differential proliferation and gene expression in response to changes in spreading. Understanding how best to vascularize engineered and native infarcted tissue is now a question of increasing importance due to a greater need for organ replacements from longer lifespans, as well as the continued rise in cardiovascular disease. This proposal aims to reveal some of the molecular mechanisms behind the environmental regulation of angiogenesis for optimal tissue vascularization.